High-speed FRET screening for optical proteomics in a microfluidic format

Viput Visitkul; Daniel R. Matthews; Gregory E. Weitsman; Melanie D. Keppler; Simon M. Ameer-Beg

doi:10.1117/12.875510

10 February 2011 High-speed FRET screening for optical proteomics in a microfluidic format

Viput Visitkul, Daniel R. Matthews, Gregory E. Weitsman, Melanie D. Keppler, Simon M. Ameer-Beg

Proceedings Volume 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX; 79020P (2011) https://doi.org/10.1117/12.875510
Event: SPIE BiOS, 2011, San Francisco, California, United States

Abstract

Cancer studies require a thorough understanding of how human gene expressions and DNA modifications are translated at the proteome level. In order to unravel the large and complex interactions between proteins, we have developed a compact lifetime-based flow cytometer, utilising a commercial microfluidic chip, to screen large non-adherent cell populations. Fluorescent signals from cells are detected using time correlated single photon counting (TCSPC) in the burst integrated fluorescence lifetime (BIFL) mode and used to determine the lifetime of each cell. Initially, the system was tested using 10 μm highly fluorescent beads to determine optical throughput and detection efficiency. The system was validated with 293T monkey kidney adenocarcinoma cell line transiently transfected with a FRET standard, consisting of eGPF and mRFP1 fluorescent proteins linked by a19 amino-acid chain. Analysis software was developed to process detected signals in BIFL mode and chronologically save the transient burst data for each cell in a multi-dimensional image file.

Citation Download Citation

Viput Visitkul, Daniel R. Matthews, Gregory E. Weitsman, Melanie D. Keppler, and Simon M. Ameer-Beg "High-speed FRET screening for optical proteomics in a microfluidic format", Proc. SPIE 7902, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues IX, 79020P (10 February 2011); https://doi.org/10.1117/12.875510

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KEYWORDS

Luminescence

Fluorescence resonance energy transfer

Microfluidics

Photons

Signal detection

Proteins

Green fluorescent protein

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